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Role of the phenylalanine 260 residue in defining product profile and alcoholytic activity of the α-amylase AmyA from Thermotoga maritima

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Abstract

Some α-amylases besides catalyzing the hydrolysis of α-1,4 glycosidic bonds in starch are also capable of carrying out some transglycosylation activity. The importance of aromatic residues near the catalytic site in determining the ratio of these two competing activities has been remarked in the past. In the present work we investigated the role of residue 260 in the product profile of the α-amylase AmyA from Thermotoga maritima. This phenylalanine residue, two positions after the glutamic acid/base catalyst was substituted by both tryptophan and glycine residues, showing opposite behaviors. The tryptophan mutant displayed a very similar product profile pattern to that of the wild-type enzyme; while the mutant Phe260Gly showed a higher transglycosylation/hydrolysis ratio. When the Phe260Trp mutation was constructed in the context of His222Gln, a mutant we have already reported with an increased transglycosylation/hydrolysis ratio and a higher alcoholysis activity, the resultant enzyme showed an apparent higher hydrolysis/transglycosylation ratio and a change to shorter products pattern than the single mutant enzyme, still maintaining the increased alcoholytic activity provided by the His222Gln mutation. The mutant Phe260Gly, on the other hand showed by itself a higher alcoholytic activity, similar to that of the His222Gln mutant.

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Abbreviations

AmyA:

α-amylase from Thermotoga maritima

CGTase:

cyclodextrin glucanotransferase

G1–G7:

oligosaccharides from G1 (glucose) through G7 (maltoheptaose)

GH:

glycoside hydrolase

HPLC:

high performance liquid chromatography

TLC:

thin layer chromatography

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Authors and Affiliations

  1. Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México

    Juanita Y. Damián-Almazo, Agustin López-Munguía, Xavier Soberón-Mainero & Gloria Saab-Rincón

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  1. Juanita Y. Damián-Almazo
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  2. Agustin López-Munguía
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  3. Xavier Soberón-Mainero
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  4. Gloria Saab-Rincón
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Correspondence to Gloria Saab-Rincón.

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Damián-Almazo, J.Y., López-Munguía, A., Soberón-Mainero, X. et al. Role of the phenylalanine 260 residue in defining product profile and alcoholytic activity of the α-amylase AmyA from Thermotoga maritima . Biologia 63, 1035–1043 (2008). https://doi.org/10.2478/s11756-008-0170-4

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